Conjugate compound comprising a macrocyclic ligand and an antibody

Description

[0001] This invention relates to a conjugate compound comprising a macrocyclic ligand and an antibody, to a metal complex of the conjugate compound and to formulations of the metal complex for use in therapy and diagnosis.

[0002] It is known to label an antibody with a metal atom, in order to target the metal atom to a specific tissue type, both in vitro and in vivo. Such labelled antibodies have applications in locating specific tissue types (e.g. employing computer-aided tomographic techniques where the metal atom is in some way detectable) and in the treatment of cell disorders (e.g. treating mammalian tumours where the metal atom is a cytotoxic radionucleide).

[0003] Conventionally, the metal atom has been complexed to a conjugate compound comprising a ligand covalently attached to an antibody. The ligand may be, for example, an acyclic chelate such as a substituted diethylenetriaminepentaacetic acid (DTPA) (Gansow O.A. et al, Inorg. Chem., (1986), 25, 2772) or ethylenediaminetetraacetic acid (EDTA) (Meares, C.F. et al, Acc. Chem. Res., (1984), 17, 202). Such acylic complexes however tend to be unstable in vivo either as a result of acid-catalysed decomplexation or competitive chelate binding by Ca²⁺ or Zn²⁺ in serum or as a result of competition from transferrin (Moerlein, S.M. et al, Int. J. Nuc. Med. Biol., (1981), 8, 277). The lack of stability can result in uncomplexed metal atoms in the body which have a cytotoxic effect on healthy tissue or which markedly reduce the signal-to-noise ratio of an imaging technique. The use of macrocyclic ligands in the labelling of antibodies has been suggested in broad terms (Gansow, O.A. et al, Am. Chem. Soc. Symp/ Ser., (1984), 241, 215; Published British patent application GB2122641A; Meares, C.F. et al, Anal. Biochem., (1985), 148, (249-253)). The attachment of metal ions to antibodies by means of acyclic and macrocyclic chelators, including aminobenzyl TETA - based macrocyclic ligands has been reviewed (Meares, C.F., in Protein Tailoring for Food and Medical Uses, (Feeny, R.E. and Whitaker, J.R., eds.), pp 339 - 352, Marcel Dekker, Inc., New York (1986)).

[0004] The object of the present invention is to provide improved conjugate compounds involving macrocylic ligands capable of binding metals to give complexes which are stable, in vivo.

Summary of the Invention

[0005] According to a first aspect of the present invention there is provided a conjugate compound having the following structure:


wherein
   R¹ is -(CH₂)p-R⁶-(CH₂)q- where p and q are the same or different and are 0, 1 or 2, and -R⁶- is -(CH2)n-, where n is 0 or 1, -NH-, -O-, -S- or


R¹ optionally being alkyl substituted, provided that neither p nor q is 0 unless R⁶ is -CH₂-,
R₂ are -CH₂CH₂- or CH₂CH₂CH₂-, optionally alkyl, alkoxyalkyl or hydroxyalkyl substituted,
R4 is:




optionally alkyl substituted, wherein R⁷ is -H, alkyl, hydroxyalkyl, or alkoxyalkyl; R⁵ is a diradical adapted to link the antibody to the macrocyclic part of the conjugate compound such that the binding affinity and specificity of the antibody are substantially unimpaired and such that the macrocyclic part of the conjugate molecule may coordinate with a metal; and Ab is an antibody.

[0006] The conjugate compound of the invention provides for complexes of metals or such as Tc, Co, Re, Cu, Au and Ag, Pb, Bi, In and Ga which are useful reagents for in vivo imaging and therapy. Particularly preferred are complexes of Tc and Cu.

[0007] R¹ is preferably -CH₂CH₂CH₂- or -CH₂CH₂-O-CH₂CH₂-. The radicals R² are preferably the same and may be -CH₂CH₂-. In each case, one or more hydrogen atoms may be replaced with an alkyl hydroxyalkyl or alkoxyalkyl radical, such as lower alkyl radical, for example methyl.

[0008] The aromatic ring, where present, in R⁴ may be 1,3,5 or 1,3,4 substituted depending to the ring size required and the synthetic route adopted to make the compound.

[0009] R⁴ may be substituted with lower alkyl such as methyl. R⁷ is preferably -H or lower alkyl (C₁-C₃) such as methyl, but may be higher alkyl such as C₄-C₁₅ alkyl.

[0010] The linker, R⁵, may be any diradical adapted to link the antibody to the macrocyclic part of the conjugate compound such that the binding affinity and specificity of the antibody are not substantially impaired and such that the macrocyclic part of the conjugate molecule may coordinate with a metal, such as a transition metal or a B-metal.

[0011] The antibody, Ab, may be a complete antibody molecule or a fragment thereof or an analogue of either of these, provided the antibody Ab comprises a specific binding region, for example a Fab′ or F(ab)₂′ fragment. The antibody Ab may be a polyclonal or a monoclonal antibody or a fragment thereof. Thus the antibody Ab may be obtained from a hybridoma cell line or other animal cell line. Preferably the antibody Ab has specificity for a mammalian tumour.

[0012] In a second aspect of the invention there is provided a conjugate compound of the invention including a complexed metal atom. The metal is preferably a metal selected from Tc, Re, Co, Cu, Au, Ag, Pb, Bi, In and Ga. Particularly preferred are complexes of Tc and Cu. The metal is suitably a radioactive isotope.

[0013] The radioisotopes of technetium (Tc) and rhenium (Re) provide useful reagents for imaging and therapy. In particular, ^99mTc is a strong γ-emitter and, as such is useful in γ-ray imaging. ^186/188Re is a good β-emitter and is useful for β-particle therapy. Both are readily available from a generator as the MO₄ anion, and may be complexed in a reduced form to provide complexes which are stable in body fluids.

[0014] In a preferred form of the first aspect of the invention there is provided a conjugate compound having the structure:


wherein R⁵ and Ab are as defined for structure I above.

[0015] In a preferred form of the second aspect of the invention there is provided a conjugate compound of structure II complexed with a technetium, copper or rhenium atom, preferably ^99mTc, ⁶⁷Cu, ⁶⁴Cu, or ^186/188Re.

[0016] In a further preferred form of the first aspect of the invention there is provided a conjugate compound having the structure:


wherein m is 1 to 5, and R⁵ and R⁷ are as defined for structure I above.

[0017] The conjugate compound of structure III is suitable for complexing a range of metal atoms, including Tc, Cu, Co, In, Au and Ag, optimum binding being achieved by selection of the ring size by adjustment of parameter m.

[0018] In a further preferred second aspect of the invention, there is provided a conjugate compound of structure III wherein m is 3 and R³ is -H or -CH₃ complexed with a technetium, rhenium, copper or gold atom, preferably ^99mTc, ⁶⁷Cu, ⁶⁴Cu ,¹⁹⁹Au, ^186/188Re.

[0019] The conjugate compound of the second aspect of the invention may be used as a pharmaceutical, for example, in the treatment of mammalian tumours or as a diagnostic reagent for use in imaging in vivo as a tracer.

[0020] In a third aspect, the invention provides a composition comprising a conjugate compound of the second aspect of the invention or an addition salt thereof and a pharmaceutically acceptable carrier. The composition may be used in the treatment of tumours or as a diagnostic reagent for imaging in vivo.

[0021] In a fourth aspect, the invention provides a compound of the following general structure:


wherein R¹, R² and R⁴ are as defined for structure I above and R⁸ comprises a reactive group covalently attached to R⁴. The reactive group provides a point of attachment for an antibody (Ab) either directly, or through a linker. R⁸ may comprise -(CH₂)_n-NH₂, -(CH₂)_n-Ar-(CH₂)_m-NH₂, -(CH₂)_n-CO₂H, -(CH₂)_n-Ar-CO₂H, -(CH₂CH₂O)_n-CH₂CH₂NH₂, -(CH₂)_n-NCS, -(CH₂)_n-Ar-NCS, or -(CH₂)_nNHCOR⁹, wherein n and m = 0 to 4 and are the same or different and R⁹ is a haloalkyl such as -CH₂Br. The isothiocyanate and haloalkyl functionalised cycles may be linked directly to an antibody thiol or amino group. Most preferably R⁸ is a primary amine such as -CH₂-Ar-CH₂NH₂.

[0022] It has been discovered that an exocyclic primary amine having a pKa from 8 to 10 exhibits exceptional qualities of selectivity allowing the attachment of linkers to the macrocycles of the invention without substantial disadvantageous side reactions and polymerisation resulting from the existence of the saturated ring nitrogens. The selectivity effect is optimised by careful pH control. Suitably, the attachment of a linker to compound IV is conducted at from pH 6 to pH 8. The primary amine may be conveniently reacted with an acylating agent, such as an activated ester moiety on a linker compound.

[0023] The linker comprises a moiety for reaction with R⁸ and a moiety for attachment to antibody. Suitably the moiety for attachment to antibody is a thiol-specific group such as a maleimide group or a vinyl group conjugated with an aromatic nitrogen heteroycle (e.g. a vinyl pyridine group).

[0024] In a preferred aspect of the invention, a primary amine group is covalently linked to a maleimide or vinyl pyridine-containing linker compound, which may be subsequently reacted with a thiol group on the antibody or modified antibody. This compound therefore provides an important intermediate in the production of a conjugate compound according to the first or second aspect of the invention.

[0025] The invention further provides addition salts of the conjugate compounds described.

[0026] The invention further provides a method for preparing conjugate compounds of the first aspect of the invention comprising reacting together a compound of the fourth aspect of the invention with a linker and an antibody or an antibody fragment.

[0027] Compounds according to the present invention can be used in a method for the treatment of a diseased cell comprising administering an effective amount of a conjugate compound according to the second aspect of the invention, the conjugate compound comprising an antibody having specificity to the diseased cells and the macrocycle including a metal cytotoxic to the diseased cells. The diseased cells may be, for example, mammalian tumour cells.

[0028] Compounds according to the present invention can be further used in a method of tumour imaging comprising administering an effective amount of a conjugate compound of the second aspect of the invention,the conjugate compound comprising an antibody to the tissue and the macrocycle including a detectable metal. The tissue may be for example, mammalian tumour cells.

[0029] The invention is illustrated by the following Examples.

Description of Specific Embodiments

Example 1

[0030] A synthesis was performed of a macrocyclic compound having the structure:

[0031] To a solution of sodium ethoxide (0.051M) in dry ethanol (100cm³) under nitrogen, was added diethyl malonate (16g, 100 mmol) in dry ethanol (35cm³) and the mixture was stirred at room temperature (0.5h). To the cooled solution was added a solution of α-bromo-paratolunitrile (10g, 0.051 mol) in dimethylformamide (50cm³) and the mixture was refluxed (24h). After cooling to room temperature, distilled water (150cm³) was added, the mixture filtered and the filtrate was extracted with di ethyl ether (3 x 50cm³). The ether was removed under reduced pressure and the residue distilled to yield p-cyanobenzyldiethylmalonate (6g, 45%), bp 150°C (0.01mm) (δ_H CDCl₃) 7.59(2H, d, Br); 7.33 (2H d, Ar); 4.16 (4H, mult, CH₂O) 3.63 (1H,t, CHCH₂), 3.27 (2H, d, CHCH₂); 1.22 (6H, t, CH₃). (δ_c CDCl₃) 167.3(COEt); 143.3, 132.1, 129.5, 116.5(Ar); 110.5 (CN); 61.6 (CH₂O); 53.0 (CO₂Et)₂); 34.4 (CH₂-Ar); 13.8(CH₃).

[0032] A mixture of 1,4,8,11-tetraazaundecane (2.77g, 17.3mmol) and p-cyanobenzyldiethylmalonate (4.75g, 17.3mmol) in dry ethanol (50cm³) was refluxed for five days, evaporated to dryness and the residue chromatographed on silica gel (0.063 0.20mm, Merck) gradient eluting with an aqueous ammonia/methanol dichloromethane solvent of composition which was initially 1:12:87 and finally 6:44:50. The required diamide was isolated as a colourless solid (1.1g, 19%), mp 209-211°C. (δ_H CDCl₃) 7.56 (2H,d,Ar), 7.33 (2H,d,ArH), 6.65 (2H, brs, NHCO), 3.50 (2H, mult, CH₂Ar), 3.25 (4H, mult., CH₂NC0), 1.78 (2H,brs,NH), 1.65 (2H, mult., CH₂C) m/e (isobutane DCI) 344 (m⁺=1) IR (KBr) ν 3290 (NH), 2910, 2805, (CH), 2225 (C=N), 1638(NCO), 1530 (NH) Rf(SiO₂: aq.NH₃/MeOH/CH₂Cl₂) (6:44:50) 0.25.

[0033] To the diamide (0.97g, 2.8 mmol) in tetrahydrofuran (25cm³) was added by syringe under nitrogen a borane-tetrahydrofuran solution (47cm³, 1.0M) and the mixture refluxed (24h). After destroying the excess borane by careful addition of methanol (4cm³), the solution was evaporated to dryness and the residue treated with hydrochloric acid (6M, 30cm³) and refluxed for 3h. On cooling, the solution was basified with sodium hydroxide (2.5cm³) and extracted with chloroform (3 x 50cm³). Removal of solvents under reduced pressure gave a colourless residue which was recrystalised from toluene to give the desired pentamine (Va) as a colourless solid (0.75g, 83%), mp 149-150°C. (δ_H CDCl₃) 7.21(2H,d,ArH), 7.14(2H,d,ArH), 3.83(2H,d,CH₂Ar) 2.8 2.4(19H, mult., CH₂Ar+ CH₂N), 2.2 (6H,brs,NH), 1.71 (2H,quint, CH₂C) m/e (isobutane DCI) 320 (m⁺=1), 319 (M⁺) (δ_c CDCl₃) 140.9 (ArCH₂-C aryl) 139.0 (CH₂-C(aryl); 129.1, 127.0 (ArCH); 55.8, 50.7, 49.3, 46.1 (CH₂N); 40.8 (CH), 38.6 (CH₂-Ar); 29.3 (CH₂).

[0034] To a solution of the p-nitrophenyl ester of 2-vinyl, 6-methoxy acetic acid pyridine (31.2mg, 0.1 mmol) in p-dioxan ( 5cm³) was added a solution of Va (31.9 mg, 0.1mmol) in buffered water (pH6.8, 5cm) 0.5M in 1,4-piperazine bis (ethanesulphonic acid) [PIPES]) and the solution stirred at 20°C for 3h. Purification of the ester by ion-exchange HPLC gave the desired ester Vb (44.5mg, 90%). m/e (fast atom bombardment, glycerol/H₂O) 495 (M⁺+1), 494.33 (M⁺), 304, 224, 185, 115. (δ_H D₂O as the diacetate salt) 7.76 (1H,t,pyCH,J 7.Hz₂); 7.48 (1H,d,pyCH); 7.30 (1H,d,pyCH); 7.17 (3H,brs,ArCH); 6.74 (1H,dd,vinylCH); 6.03(1H,d, J 17.7); 5,50 (1H,d,J 11.0); 4.64 (2H, COCH₂O); 4.33 (2H₃S₃ CH₂O); 4.14 (2H,S, CH₂NH); 2.98-2.32 (19H, mult. CH₂N+CH₂Ar+CHCH₂), 1.84 (2H, mult, CH₂CH₂CH₂).

[0035] The maleimide functionalised macrocycle Vc may be made in a similar manner to that described for Vb, using the p-nitrophenyl or N-hydroxysuccinimide ester of N-(4-carboxycyclohexylemthl)-malemide, (Yamada, et al, Eur. J. Biochem, (1979), 101, 395).

[0036] In order to link the functionalised macrocycle to the antibody, the antibody was first reacted with Traut's reagent (2-iminothiolane) to give free thiol groups for attachment. For example, a solution of Lym-1 (2mg) in 0.2M sodium phosphate buffer (pH 7.4) was mixed with 75 µdm³ of 2-iminothiolane (20mM) in 50 mM triethanolamine-HCl (pH 8) and B-thioethanol (6 µdm³) was added. After incubating at 4°C (1h), the modified antibody was purified on a G-10 Sepharose column. To a solution of the antibody at pH 6.8 (PIPES buffer) was added the vinyl-pyridine conjugated macrocycle, (Vb), and the reaction was allowed to proceed for 3 hours at room temperature. The resulting macrocycle-linker-antibody conjugate was purified on a G-10 Sepharose column.

[0037] Complexation of Vb or Vc with technetium was effected using modifications of the reported literature methods (Troutner et al Int. J. Appl. Radiat Isot. (1982), 33, 891; Childs R.L. et al , J. Nucl. Med., (1985), 26, 193) using TcO₄⁻ in the presence of stannous tartrate and using a phosphate or succinate buffer, in order to facilitate transfer of ^99mTc from reduced technetium (V) to the macrocycle.

[0038] Complexation of rhenium was achieved by reaction of equimolar quantities of Vb and ReOCl₃ (PPH₃)₂ (Johnson et al, J. Chem. Soc., (1964), 1054) in chloroform. After 5 min. at 25°C, diethylether was added and a ReOCl(Vb)2⁺ complex precipitated. In aqueous solution this exists primarily as the ReO₂(Vb)⁺ complex, as the chlorine trans to the Re-oxo bond is labile.

[0039] A solution of ⁶⁴CuCl₂ (30µl), was added to the macrocycle conjugate [200µl with approx. 3mg Ab/ml] in 0.1M sodium acetate or 0.1M sodium succinate at a pH of 6.0. The solution was incubated at 35°C for 0.5 h, and then chromatographed on a Sephadex G-50 (fine) gel column [which had been previously swollen (overnight) in the PBS buffer used for protein eluting] of dimension 1cm x 6cm, collecting 0.5ml aliquots and counting each fraction in order to separate the desired copper-bound antibody macrocycle complex.

Example 2

[0040] A synthesis was performed of a macrocyclic compound having the structure:

[0041] 6- Cyanocoumarin (3.46g, 20mmol), prepared from 6-aminocoumarin via diazotisation followed by reaction with cuprous cyanide (Morgan E.T., et al, J. Chem. Soc. (1904), 1230), was added to a solution of 1,9-diamino-3, 7-diazanonane (3.20g, 20mmol) in methanol (200cm³) and the mixture was refluxed under argon for 6 days following the method of Kimura (J. Chem. Soc. Commun. (1985), 335). After evaporation of the methanol, the residue was chromatographed on silica as described in Example 1, and the resultant amide was reduced using excess borane-tetrahydrofuran to give VI which was purified by recrystallisation from hot toluene, or using reverse phase HPLC methods.

[0042] The macrocycle was linked to a maleimide or a vinyl-pyridine linker as described in Example 1, and the functionalised macrocycle linked to an antibody Fab′ or F(ab)₂′ fragment as described above. Complexation of technetium or rhenium (V) was achieved under reducing conditions from the corresponding salt, using the methods disclosed in Example 1.

Example 3

[0043] A synthesis was performed of a macrocyclic compound having the structure:

[0044] Sodium metal (115mg, 5mmol) was dissolved in dry methanol (100cm³) and N, N¹-3,7-tetra(p-toluenesulphonyl)-3,7-diazanonane-1,9 diamine (1.95g, 2.51 mmol) was added as a solid. The mixture was refluxed under nitrogen for 1 hour, and solvent was removed to yield a solid disodium salt. After removing traces of methanol in vacuo, the solid was dissolved in dry dimethylformamide (50cm³) and the solution was heated to 80°C. A solution of 6,6-dibromemethyl-3-nitrobenzine (747mg, 2.5 mmol) in dimethylformamide (20cm³) was added over a period of 1 hour. After stirring for 2 hours at 80°C, water (40cm³) was added over a period of 15 minutes and the mixture was stirred for 16 hours at 20°C. The resulting oily product was separated, dissolved in dichloromethane (50cm³), washed with water (3x15cm³) and the solvent was evaporated. The cyclic tetratosylate was purified by precipitation from CH₂Cl₂hexane (1:4 v/v) and subsequently by chromatography on a neutral column eluting with dichloromethane/methanol, (1%), to yield a tosylated macrocycle as a colourless glass. Detosylation was effected by adding phenol (2g) and hydrogen bromide in acetic acid (45%, 100cm³) to the macrocycle and stirring for 24 hours at 80°C. The resulting mixture was cooled and the solvent was removed under reduced pressure, adding toluene to assist the removal of the acetic acid. The residue was dissolved in water (100cm³) and washed with dichloromethane (4x50cm³). The aqueous phase was evaporated and the product was redissolved in water (100cm³) and passed down an anion exchange resin (OH⁻form, Amberlite IR400). The aqueous solution thus obtained was evaporated, leaving a product which was crystallised as the hydrochloride salt from ethanol/hydrochloric acid. The amine was regenerated by passage through an anion exchange column, and is a pale yellow oil. Reduction of the aromatic nitro group to the corresponding amino group was effected by dissolving the free amine (618mg, 2 mmol) in ethanol, adding palladium on carbon (100mg, 10%) and hydrogenating under 2 atmospheres of -30°C for 3 hours. After filtering to remove spent catalyst, the solvent was removed to yield compound VI which was stored at ,30°C under nitrogen.

[0045] Complexation of Re was achieved as described in Example 1.

Example 4

[0046] A synthesis was performed of a macrocyclic compound having the structure:

[0047] 6-nitrocoumarin (9.55g, 50 mmol) was added to a solution 1,9-diamino-3,7-diazanonane (8g, 50 mmol) in methanol (200cm³) under argon, and the mixture was refluxed for 6 days. After evaporation of the methanol, the residue was chromatographed on silica, eluted with CH₂Cl₂/CH₃OH/NH₄OH (70:27:3 v/v) to yield a yellow oil which was further purified by recrystallisation from ethanol/hydrochloric acid to give a trihydrochloride salt of the cyclic amide. Successive reduction of the amide with borane-dimethylsulphide, O-methylation of the phenolic -OH group (under nitrogen) and reduction of the aromatic nitro group using palladium on carbon as described in Example 3 above resulted in macrocycle IX.

[0048] Complexation of technetium was effected using techniques known in the literature employing (NH₄)TcO₄ and the macrocycle in the presence of Na₂S₂O₄ or stannous tartrate.

[0049] The macrocycle produced in Example 1, 2, or 3 may be covalently bound to an antibody or an antibody fragment, using standard techniques, before or after complexation with the metal. For example a Fab′-linker macrocycle conjugate according to the present invention may be prepared substantially as described below for the model macrocyle, 14-(5-aminophenyl)-1, 4, 8, 11-tetraazocyclotetradecane.
14-(5-aminophenyl)-1, 4, 8, 11-tetraazocyclotetra decane (1m mole) was dissolved in 0.2M phosphate buffer (pH 7.4) and treated with the heterobifunctional cross-linking reagent succinimidyl-4-(p-maleimidophenyl) butyrate (1m mole) dissolved in 0.2M phosphate buffer (pH 7.4), for two hours at room temperature. The resulting macrocycle-linker was purified using silica gel column chromatography, substantially as decribed by Kimura et al, (Jnl. Chem. Soc. Chem. Comm., (1985), p 385).

[0050] A monoclonal antibody was treated with the enzyme pepsin to generate a F(ab′)₂ fragment. The F(ab′)₂ fragment was then purified using gel permeation chromatogrpahy. The purified F(ab′)₂ fragment was then treated with the reducing agent dithiothreitol (1m M) at pH 8 for three hours at room temperature. The resulting Fab′ fragments were then acidifed and passed through a G.10 Sepharose column to yield pure Fab′ fragments. The resulting Fab′ (0.1m mole) was then dissolved in 0.2M phosphate buffer (pH 7.4) and added to a solution of the macrocycle-linker conjugate (1m mole) in 0.2M phosphate buffer (pH 7.4).

[0051] The Fab′ fragment and macrocycle-linker conjugate were then allowed to react for three hours under argon at room temperature and the resulting macrocycle-linker-Fab′ conjugate purified on a G.10 Sepharose column.

Claims

1. A conjugate compound having the following structure:

wherein
   R¹ is -(CH₂)p-R⁶-(CH₂)q- where p and q are the same or different and are 0, 1 or 2, and -R⁶- is -(CH₂)n-, where n is 0 or 1, -NH-, -O-, -S- or

R¹ optionally being alkyl substituted, provided that neither p nor q is 0 unless R⁶ is -CH₂-,
   R² are -CH₂CH₂ - or -CH₂CH₂CH₂ -, optionally alkyl, alkoxyalkyl or hydroxyalkyl substituted,
   R⁴ is



optionally alkyl substituted, wherein R⁷ is -H, alkyl, hydroxyalkyl, or alkoxyalkyl
   R⁵ is a diradical adapted to link the antibody to the macrocycle part of the conjugate compound such that the binding affinity and specificity of the antibody are substantially unimpaired and such that the macrocyclic part of the conjugate molecule may coordinate with a metal, and Ab is an antibody.

2. A conjugate compound according to claim 1 wherein R¹ is -CH₂CH₂CH₂-.

3. A conjugate compound according to claim 1 wherein R¹ is -CH₂CH₂OCH₂CH₂-.

4. A conjugate compound according to claim 1 wherein R² are the same and are -CH₂CH₂-.

5. A conjugate compound according to claim 1 wherein R⁷ is -H or C₁ to C₃ alkyl.

6. A conjugate compound having the structure:

wherein R₅ and Ab are as defined in claim 1.

7. A conjugate compound according to claim 1 having the structure:

wherein m is 1 to 5 and R⁵ and R⁷ are as defined in claim 1.

8. A conjugate compound according to claim 1, including a complexed metal atom.

9. A conjugate compound according to claim 8 wherein the complexed metal atom is selected from Tc, Re, Co, Cu, Au and Ag.

10. A conjugate according to any one of the preceding claims including a complexed Tc or Cu atom.

11. A composition comprising a conjugate compound as defined in claim 1, or an addition salt thereof, and a pharmaceutically acceptable carrier.

12. A compound having the structure:

wherein R¹, R² and R⁴ are as defined in claim 1 and R⁸ comprises an exocyclic amine having a pKa from 8 to 10 covalently attached to R⁴.

13. A compound according to claim 12 wherein R⁸ is -CH₂-Ar -CH₂NH₂.

14. A method for preparing a conjugate compound as defined in claim 1 comprising reacting together a compound as defined in claim 12 with a linker and an antibody.

Ansprüche

1. Konjugatverbindung der folgenden Struktur

in welcher
   R¹ für -(CH₂)p-R⁶-(CH₂)q- steht, worin p und q gleich oder verschieden sind und 0, 1 oder 2 bedeuten und -R⁶- für (CH₂)n-, worin n 0 oder 1 darstellt, -NH-, -O-, -S- oder

steht, wobei R¹ gegebenenfalls alkylsubstituiert ist, mit der Maßgabe, daß weder p noch q für 0 steht, sofern R⁶ nicht -CH₂- bedeutet,
   R² für -CH₂CH₂- oder -CH₂CH₂CH₂- steht, die gegebenenfalls alkyl-, alkoxyalkyl- oder hydroxyalkylsubstituiert sind,
   R⁴



bedeutet, die gegebenenfalls alkylsubstituiert sind, wobei R⁷ -H, Alkyl, Hydroxyalkyl oder Alkoxyalkyl darstellt,
   R⁵ ein zweiwertiger Rest ist, der zur Bindung des Antikörpers an den Makrozyklus-Teil der Konjugatverbindung so befähigt ist, daß Bindungsaffinität und -spezifität des Antikörpers im wesentlichen unbeeinträchtigt sind und der Makrozyklus-Teil des Konjugatmoleküls sich mit einem Metall koordinieren kann, und Ab für einen Antikörper steht.

2. Konjugatverbindung nach Anspruch 1, in welcher R¹ für -CH₂CH₂CH₂- steht.

3. Konjugatverbindung nach Anspruch 1, in welcher R¹ für -CH₂CH₂OCH₂CH₂- steht.

4. Konjugatverbindung nach Anspruch 1, in welcher die Reste R² gleich sind und für -CH₂CH₂- stehen.

5. Konjugatverbindung nach Anspruch 1, in welcher R⁷ für -H oder C₁-C₃-Alkyl steht.

6. Konjugatverbindung der Struktur

in welcher R₅ und Ab die in Anspruch 1 angegebene Bedeutung haben.

7. Konjugatverbindung nach Anspruch 1 der Struktur

in welcher m für 1 bis 5 steht und R⁶ und R⁷ die in Anspruch 1 genannte Bedeutung haben.

8. Konjugatverbindung nach Anspruch 1, die ein komplexiertes Metallatom enthält.

9. Konjugatverbindung nach Anspruch 8, in welcher das komplexierte Metallatom ausgewählt ist aus Tc, Re, Co, Cu, Au und Ag.

10. Konjugat nach einem der vorhergehenden Ansprüche, das ein komplexiertes Tc- oder Cu-Atom enthält.

11. Zusammensetzung, die eine Konjugatverbindung nach Anspruch 1 oder ein Additionssalz derselben und einen pharmazeutisch verwendbaren Träger enthält.

12. Verbindung der Struktur

in welcher R¹, R² und R⁴ die in Anspruch 1 genannte Bedeutung haben und R⁶ ein exozyklisches Amin mit einem pKa von 8 bis 10 kovalent an R⁴ gebunden enthält.

13. Verbindung nach Anspruch 12, in welcher R⁸ für -CH₂-Ar-CH₂NH₂ steht.

14. Verfahren zur Herstellung einer Konjugatverbindung nach Anspruch 1, bei welchem eine Verbindung nach Anspruch 12 mit einem Linker oder einem Antikörper zur Reaktion gebracht wird.

Revendications

1. Composé conjugué ayant la structure suivante

dans laquelle
   R¹ représente -(CH₂)_p-R⁶-(CH₂)_q- , où p et q sont égaux ou différents et valent chacun 0, 1 ou 2 , et -R⁶- représente -(CH₂) - (où n vaut 0 ou 1 ), -NH- , -O- , -S- ou

R¹ pouvant éventuellement comporter un substituant alkyle , à la condition que ni p ni q ne soient nuls sauf si R⁶ représente -CH₂- ,
   les R² représentent -CH₂CH₂- ou -CH₂CH₂CH₂ - ,portant éventuellement comme substituant un groupe alkyle , alcoxyalkyle ou hydroxyalkyle ,
   R⁴ représente



éventuellement substitués par un groupe alkyle , et où R⁷ représente H ou un groupe alkyle , hydroxyalkyle ou alcoxyalkyle ;
   R⁵ est un diradical convenant pour lier l'anticorps à la partie macrocyclique du composé conjugué , de manière à ne sensiblement pas altérer l'affinité de liaison et la spécificité de l'anticorps et que la partie macrocyclique de la molécule du conjugué puisse former un coordinat avec un métal, et Ab représente un anticorps .

2. Composé conjugué selon la revendication 1 , dans lequel R¹ représente -CH₂CH₂CH₂- .

3. Composé conjugué selon la revendication 1 , dans lequel R¹ représente -CH₂CH₂OCH₂CH₂- .

4. Composé conjugué selon la revendication 1 , dans lequel les R² sont identqiues et représentent chacun un groupe -CH₂CH₂- .

5. Composé conjugué selon la revendication 1 , dans lequel R⁷ représente -H ou un groupe alkyle en C₁ à C₃ .

6. Composé conjugué ayant la structure :

dans laquelle R₅ et Ab sont tels que définis à la revendication 1 .

7. Composé conjugué selon la revendication 1 ,ayant la structure

dans laquelle m vaut 1 à 5 , et R⁵ et R⁷ sont tels que définis à la revendication 1 .

8. Composé conjugué selon la revendication 1 , comprenant un atome de métal complexé .

9. Composé conjugué selon la revendication 8 , dans lequel l'atome de métal complexé est choisi parmi Tc , Re , Co , Cu , Au et Ag .

10. Conjugué selon l'une quelconque des revendications précedentes , comprenant un atome de Tc ou Cu complexé .

11. Composition comprenant un composé conjugué,tel que défini à la revendication 1 , ou un sel d'addition de ce composé , et un excipient pharmaceutiquement acceptable .

12. Composé ayant la structure

dans laquelle R¹ , R² et R⁴ sont tels que définis à la revendication 1 , et R⁸ comprend une amine exocyclique,ayant un pKa de 8 à 10 , fixée par covalence sur R⁴ .

13. Composé selon la revendication 12 , dans lequel R⁸ représente un groupe -CH₂- Ar -CH₂NH₂ .

14. Procédé pour préparer un composé conjugué tel que défini à la revendication 1 , comprenant la réaction d'un composé tel que défini à la revendication 12 avec un agent de liaison et un anticorps .